RESEARCH ARTICLE 2049

Development 138, 2049-2057 (2011) doi:10.1242/dev.061176 © 2011. Published by The Company of Biologists Ltd Transcription precedes loss of Xist coating and depletion of H3K27me3 during X- reprogramming in the mouse inner cell mass Lucy H. Williams, Sundeep Kalantry*, Joshua Starmer and Terry Magnuson†

SUMMARY Repression of Xist RNA expression is considered a prerequisite to reversal of X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. We analyzed X-chromosome activity in developing ICMs and show that reactivation of expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes in the ICM that operates independently of loss of Xist RNA and H3K27me3 from the imprinted inactive-X.

KEY WORDS: X inactivation, Epigenetic reprogramming, Pre-implantation mouse development

INTRODUCTION gradually coats the Xp during pre-implantation development X-chromosome inactivation (XCI) results in the transcriptional (Huynh and Lee, 2003; Okamoto et al., 2004). By the early silencing of most genes on one of the two X- to blastocyst stage, prior to specification of extra-embryonic and equalize X-linked gene dosage in female cells with that in XY male embryonic lineages, Xist accumulation on the Xp (Xp-Xist coating) cells (Avner and Heard, 2001). Although the exact mechanism for is evident in all cells of the embryo (Sheardown et al., 1997). At X-linked gene silencing is unknown, XCI requires expression of the late blastocyst stage, when extra-embryonic and embryonic the long non-coding RNA X inactive-specific transcript (Xist) lineages are determined, Xp-Xist coating is maintained in the extra- (Brown et al., 1991; Marahrens et al., 1997; Penny et al., 1996). embryonic trophectoderm and primitive endoderm. By contrast, Xist RNA is expressed from and preferentially coats the inactive X- Xp-Xist coating is lost in epiblast cells, which define the progenitor chromosome (Clemson et al., 1996). Xist coating is followed by cells of the embryo proper (Mak et al., 2004; Okamoto et al., 2004; recruitment of multiple chromatin-modifying complexes, such as Sheardown et al., 1997). the polycomb repressive complex 2 (PRC2), which trimethylates Based on patterns of Xp-Xist coating in the blastocyst, it is lysine 27 on histone H3 to give H3K27me3 (Plath et al., 2002; assumed that all cells of the pre-implantation embryo first undergo Silva et al., 2003). H3K27me3 is a chromatin modification the imprinted form of XCI and that this imprint is maintained in the associated with gene repression, and H3K27me3 enrichment on the extra-embryonic lineages. In the developing ICM, epiblast inactive-X is essential to maintain imprinted paternal-X silencing precursor cells reverse imprinted XCI in order to subsequently in extra-embryonic lineages (Kalantry et al., 2006; Simon and initiate random XCI later in development (Mak et al., 2004; Kingston, 2009). Together, Xist coating and H3K27me3 are Okamoto et al., 2004). However, three recent reports analyzed Xp- necessary to establish stable and heritable XCI; thus, these linked gene transcription in single cells of pre-implantation epigenetic modifications are considered to be markers of X-linked embryos. Each report independently confirmed that some degree gene silencing. of Xp-linked gene silencing is evident as early as the four-cell Two types of XCI are evident during development of the female cleavage stage. Yet, despite Xist coating the Xp in all cells, Xp- mouse embryo. The first type, imprinted XCI, results in exclusive linked gene silencing is not evident in all cells of the blastocyst, silencing of the paternal X-chromosome (Takagi and Sasaki, 1975; indicating that imprinted XCI is incomplete during pre- West et al., 1977). By contrast, the second type of XCI is termed implantation development (Kalantry et al., 2009; Namekawa et al., random because either maternal-X (Xm) or paternal-X (Xp) can 2010; Patrat et al., 2009). Given these results, it is still unclear be chosen for silencing (Lyon, 1961). During early mouse whether epiblast progenitor cells, which are specified by the embryogenesis, Xist expression is first evident at the two-cell stage blastocyst stage, exhibit imprinted Xp-linked gene silencing. and occurs exclusively from the Xp (Kay et al., 1993). Xist RNA Here, we utilized RNA fluorescence in situ hybridization (FISH) techniques to measure directly the kinetics of X-linked gene Department of Genetics, Carolina Center for Genome Sciences, and Lineberger expression, as opposed to chromosome-wide markers of Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599- inactivation, in individual nuclei of developing ICMs. We provide 7264, USA. evidence that supports previous assumptions that, prior to epiblast *Present address: Department of Human Genetics, 1241 E. Catherine St, University specification, ICM cells exhibit imprinted XCI. We also make the of Michigan Medical School, Ann Arbor, MI 48109-5618, USA surprising observation that reactivation in the ICM occurs earlier †Author for correspondence ([email protected]) than previously indicated by loss of Xist coating (Mak et al., 2004;

Accepted 17 February 2011 Okamoto et al., 2004). Coincident with specification of the Nanog- DEVELOPMENT 2050 RESEARCH ARTICLE Development 138 (10) expressing epiblast lineage, but prior to loss of the Xist domain and extra-embryonic endoderm (XEN) cell lines, respectively. For combined H3K27me3 enrichment on the Xp, mid-stage ICMs exhibit an immunostaining of H3K27me3 and RNA FISH, RNA FISH was performed increase in Xp-linked gene expression, indicative of active after immunostaining. transcription. Recent results suggest that pluripotency-associated Statistical analysis of RNA FISH transcription factors NANOG, OCT3/4 (POU5F1 – Mouse RNA FISH nuclei were classified as biallelic, monoallelic or nonscorable. Genome Informatics) and SOX2 might directly regulate X- Nuclei were classified as nonscorable when no signal could be detected. chromosome activity by preventing upregulation of Xist expression For each X-linked gene, the total number of the three classes of nuclei was in mouse embryonic stem cells (ES cells) (Navarro et al., 2008). compared between early and mid-stage ICMs. Binomial exact test was used We show that forced upregulation of NANOG results in loss of Xp- to verify that nonscorable data between stages were equivalent. Fisher’s Xist coating without affecting the kinetics of Xp-linked gene exact test compared the three categories between early and mid-stage reactivation. These observations argue against a direct role for ICMs. P values <0.01 were considered to be statistically significant. The procedure was repeated for the Cot-1 RNA FISH experiments except nuclei NANOG-dependent Xist repression as a requirement for initiation were scored as either excluded, overlapping or nonscorable. of Xp expression. We present an alternative hypothesis that reversal of imprinted X-chromosome inactivation occurs by separable steps, Microscopy with gene reactivation being independent from loss of the Xist and Images for RNA FISH were obtained using a Leica DML fluorescence H3K27me3 domains. microscope with a Q-imaging Retiga 200R camera and Q-capture software. Images were processed using SPOT RT Software (Diagnostic Instruments). MATERIALS AND METHODS For Cot-1 RNA FISH and immunostaining, a Zeiss710 confocal microscope was used. Confocal sections were analyzed using Zen software Embryo collection and immunosurgery to determine overlap between signals. To determine relative intensity Embryos were collected from natural mating of either Mus musculus values obtained in the Cot-1 RNA experiments, we generated line traces domesticus CD1 females (RNA FISH) or Mus musculus molossinus JF1 across nuclei using NIH Image J software and used the plot profile tool to females (RT-PCR) to CD1 males carrying the X-GFP (D4.EGFP) transgene measure the intensity across the line traces. (Hadjantonakis et al., 1998). Female blastocysts were distinguished from –/– males by GFP expression. The Grb2 (Grb2tm2Paw) line was described RT-PCR previously (Cheng et al., 1998). Mice were exposed to light daily between RT-PCR was performed as described previously (Kalantry et al., 2009). In 06.00 h and 18.00 h. The morning of the day the plug was detected was brief, mRNA was prepped using the Dynabeads mRNA DIRECT Micro considered to be embryonic day (E)0.5. Early blastocysts were collected Kit (Invitrogen, 610.21). ICMs were lysed in 100 l of lysis/binding buffer. between E2.75 and E3.0 (~66-72 hours post-copulation). Mid-stage For RT-PCR, SuperScript III One-Step RT-PCR Platinum Taq (Invitrogen, blastocysts were collected between E3.5 and E3.75 (~84-90 hours post- 12574-035) was used to amplify the cDNA. A portion of the final RT-PCR copulation). Because embryos collected at the same time varied in product was used to perform one round of amplification in the presence of developmental stage, blastocysts were also staged according to size (a a trace amount of 32P-dCTP (Perkin Elmer, BLU513H). The final PCR measure of total cell number) and blastoceol cavity expansion (see Fig. S6 product was digested with the appropriate restriction enzyme, run on a 10% in the supplementary material). Immunosurgery procedures were acrylamide gel and exposed to film. All RT-PCR primers were described performed as described previously (Solter and Knowles, 1975). previously (Kalantry et al., 2009). Band intensities were quantified using Adobe Photoshop CS3 Extended software. Percent paternal expression was Immunostaining calculated as a percentage of total intensity of combined maternal- and Embryos were fixed in 3% paraformaldehyde for 10 minutes at room paternal-specific bands, i.e. % Xp  [Xp/(Xm+Xp)]ϫ100. In the case of ϫ temperature. After washing in 1 PBS, embryos were permeabilized in multiple maternal- or paternal-specific bands, the band intensities were 0.5% Triton X-100 for 15 minutes. Embryos were then washed in 1ϫPBS added to yield a single value. The average % Xp was compared between and blocked in 10% serum for 1 hour at room temperature. Primary early and mid-stage ICMs. P-values were calculated using two-tailed t-tests antibodies were: Nanog (Chemicon, 1:1000), Gata6 (R&D Systems, where the null hypothesis is that the mean % Xp of early and mid-stage 1:1000), H3K27me3 (Upstate, 1:250) and EED (Arie Otte, Slater Institute, samples are the same. The Netherlands, 1:200). Primary antibody incubations were performed overnight at 4°C. Embryos were washed in 1ϫPBS and incubated in secondary antibody for 45 minutes at room temperature. Secondary RESULTS antibodies coupled to Alexa Flour 594 (Invitrogen A-11037) and Alexa Imprinted XCI is established in early ICMs prior to Fluor 488 (Invitrogen A-11039) were diluted to 1:500. The embryos were epiblast specification ϫ washed in 1 PBS and mounted into 0.12 mm deep Secure-Seal spacer To investigate Xp activity in the ICM, we analyzed transcription of wells (Invitrogen, S24737) on microscope slides in Vectashield (Vector X-linked genes by RNA FISH in ICMs isolated by immunosurgery Labs) diluted 1:1 with DAPI. (Solter and Knowles, 1975). We assayed five X-linked genes RNA FISH [Rnf12 (Rlim – Mouse Genome Informatics), Atp7a, Abcb7, Ube1x FISH probes were generated from BAC and FOSMID clones (BACPAC (Uba1 – Mouse Genome Informatics) and Mecp2] that are Resources Center, CA, USA) and labeled using the Bioprime Labeling Kit expressed at robust levels during the cleavage stages of pre- (Invitrogen) with FITC-dUTP (Roche), Cy3-dCTP (GE Healthcare) implantation development (Kalantry et al., 2009; Patrat et al., or Cy5-dCTP (GE Healthcare). FOSMIDs and BACs used were: 2009). Previous allele-specific RT-PCR and RNA FISH data G135P65743A11 (Ube1x), G135P69277C4 (Mecp2), G135P63425C4 showed a bias towards Xm expression of these genes at the morula (Xist/Tsix), G135P605237C7 (Rnf12), RP24-274B9 (Abcb7), stage, indicating that these genes are subject to imprinted XCI G135P603627A9 (Smcx), G135P60684B7 (Nanog) and RP24329F11 (Huynh and Lee, 2003; Kalantry et al., 2009). We assayed (Gata6). Two FOSMIDs G135P62497G3 and G135P64951A11 were used to detect Atp7a. Mouse Cot-1 DNA (Invitrogen) was used in the Cot-1 expression of the five X-linked genes together with Xist RNA, RNA FISH experiments. RNA FISH was performed as described which marks the Xp, in ICMs of early stage blastocysts (~32-cell previously (Kalantry et al., 2009). All RNA FISH probes used to detect X- stage embryo) (Fig. 1A). If the Xp-allele of the five genes assayed linked genes were tested in trophoblast (TS) and embryonic stem (ES) cell is silenced, RNA FISH will yield a single monoallelic gene signal lines to determine detection of X-linked sequences with expected indicative of transcription from the Xm. If the Xp-linked gene is

expression patterns. Nanog and Gata6 FISH probes were tested in ES and not silenced, our assay will detect gene expression from both X- DEVELOPMENT X reactivation precedes loss of Xist RESEARCH ARTICLE 2051

Fig. 1. Mid-stage inner cell masses (ICMs) exhibit increasing Xp-linked gene expression. (A)A representative ICM assayed for Atp7a (red, left panel) and Xist and Tsix (green, right panel) by RNA fluorescence in situ hybridization (FISH). The Xist probe detects Xist RNA and the anti-sense transcript detects Tsix. Xist marks the Xp as a cloud at this stage. In ICMs, Tsix is detected in some nuclei as a pinpoint signal from the Xm (Lee et al., 1999). In the merge image, all nuclei exhibit monoallelic Atp7a expression, localized away from the Xp-Xist coat (green domains). A single nucleus is highlighted in the right corner of each image. (B)Distributions of biallelic (black), monoallelic (gray) and nonscorable (white) nuclei are presented as the average percentage of nuclei per early or mid-stage ICM. In mid-stage ICMs there is a statistically significant (P<0.01) increase in biallelism and decrease in monoallelism compared with early stage ICMs. (C)A representative nucleus illustrates biallelic Abcb7 (red, first panel) expression. H3K27me3 accumulates on the Xp above genomic levels (cyan, second panel). The H3K27me3 enrichment domain colocalizes with Xp- Xist coating (green cloud, third panel). The merge image (fourth panel) indicates that one of the two Abcb7 RNA FISH signals (marked by an arrowhead) colocalizes with both the Xp Xist and H3K27me3 domains. Nuclei are stained with DAPI. chromosomes. As a positive control, we measured expression of EED, and H3K27me3 on the Xp (Mak et al., 2004; Okamoto et al., Smcx (Kdm5c – Mouse Genome Informatics), a gene known to 2004; Silva et al., 2009). To investigate the timing of Xp escape imprinted XCI (Agulnik et al., 1994; Plusa et al., 2008). As reactivation, we isolated ICMs from mid-stage female blastocysts expected, Smcx exhibited 97% biallelic expression in cells of early (~32- to 64-cell stage embryo). We first performed dual ICMs, demonstrating that our assay is capable of detecting biallelic immunofluorescence to detect NANOG, which marks the ICM, X-linked gene transcription (see Fig. S1A in the supplementary and EED in mid-stage blastocysts. On average, 90% of NANOG material). By contrast, Rnf12, Atp7a, Abcb7, Ube1x and Mecp2 positive nuclei exhibited an EED Xp domain (Mak et al., 2004; demonstrated an average low level of biallelism, ranging from 5% Okamoto et al., 2004; Silva et al., 2009) (see Fig. S2A in the to 23% of cells per ICM (Fig. 1B). This low level of biallelism is supplementary material). Xp EED domains in ICM nuclei also comparable to the level of biallelism in trophectoderm nuclei (see exhibited accumulation of H3K27me3 (see Fig. S2B in the Fig. S1B in the supplementary material). A majority (60%) of all supplementary material). As H3K27me3 is a hallmark of early ICMs analyzed exhibited either one or no biallelic nuclei, and transcriptionally inactive chromatin domains, these data imply Xp- a number of individual ICMs exhibited exclusive (100%) linked genes are inactive in mid-stage ICMs. We next investigated monoallelic Rnf12, Atp7a, Abcb7 and Ube1x expression in every the transcriptional status of the Xp-linked genes directly using nucleus. Together, these data argue against the possibility that RNA FISH. Assaying the same five X-linked genes that previously escape from imprinted XCI is a general characteristic of ICM showed predominantly monoallelic expression in early ICMs, we nuclei and suggest that early ICMs undergo Xp silencing to the detected biallelic expression in 35-50% of cells from mid-stage same degree as do cells of the trophectoderm. Therefore, the ICMs (Fig. 1B). Importantly, in all cases of biallelism one of the majority of nuclei in the pre-implantation embryo exhibit Xp- two X-linked gene signals colocalized with Xp-Xist and linked gene silencing and undergo imprinted XCI prior to H3K27me3 accumulation, and the percentage of nuclei exhibiting specification of the embryonic lineage. Xp-Xist coating did not change significantly (Fig. 1C; see Fig. S2C in the supplementary material). In contrast to early ICMs, we never Xp reactivation occurs in the presence of the Xp- observed a mid-stage ICM that exhibited 100% monoallelic nuclei. Xist domain For all five X-linked genes analyzed, we observed a statistically Reactivation of the Xp has been described to occur in the epiblast significant increase in biallelism and a corresponding decrease in of late (fully expanded or hatched) blastocysts (~128-cell stage monoallelism (P<0.01; Fig. 1B; see Table S1 in the supplementary

embryo) after the sequential loss of Xist coating, the PRC2 subunit material). Given the cell division timing (~11.5 hours) of ICM DEVELOPMENT 2052 RESEARCH ARTICLE Development 138 (10)

Fig. 2. Dynamics of X-linked gene expression assayed by allele-specific RT-PCR. F1 hybrid mouse embryos were generated by mating JF1 females to CD1 males. Xm and Xp transcripts were distinguished utilizing single nucleotide polymorphisms between the two strains. (A)Allele- specific expression was analyzed for Xist and the X-linked genes Ube1x, Rnf12, Pdha1 and Ddx3x. Lane 1, Xm allele; Lane 2, Xp allele; Lane 3, equal amounts of Xm and Xp allele; Lanes 4-6, early F1 hybrid inner cell mass (ICM) samples; Lanes 7-9, mid-stage F1 hybrid ICM samples. Analysis of Xist expression illustrates that in both early and mid-stage ICMs, Xist is expressed from the Xp. In early ICMs, X-linked gene expression for Ube1x, Rnf12, Pdha1 and Ddx3x is largely from the Xm allele. In mid-stage ICMs there is increasing Xp expression of Xp-linked genes. (B)Average Xm and Xp contributions were calculated as a percentage of total expression (Xm+Xp). There is a statistically significant increase in average Xp expression in mid-stage ICMs compared with early stage ICMs for each X-linked gene analyzed (P<0.01). The number of ICMs examined is presented in the table. nuclei and the low level of biallelism detected at the early stage, it Fig. 2B; see Table S2 in the supplementary material). These data is also unlikely that escape from imprinted XCI, for any of the illustrate the detection of not only nascent transcripts but also genes assayed, can account for the increase in biallelic nuclei spliced polyadenylated transcripts, confirming an increase in Xp- (Bischoff et al., 2008). These data suggest that reactivation of genic linked gene expression in mid-stage ICMs. transcription occurs earlier than determined previously by analysis Together, our RNA FISH and allele-specific RT-PCR results of inactive-X epigenetic markers and, remarkably, occurs in the show that reactivation of seven Xp-linked genes occurs in the presence of Xp-Xist coating and H3K27me3 accumulation on the presence of Xp-Xist coating. In order to determine the broader Xp. transcriptional status of the Xp, we next hybridized with a Cot-1 To confirm our RNA FISH results, we analyzed allele-specific DNA probe and Xist probe in our RNA FISH experiments. Under X-linked gene expression using RT-PCR in individual ICMs RNA FISH conditions, Cot-1 DNA hybridizes to transcribed obtained from crosses between M. domesticus (CD1) and M. repetitive sequences of the genome (Hall et al., 2002). In molossinus (JF1) strains. We first measured expression of Xist combination with a Xist probe, Cot-1 RNA FISH allows RNA. In early F1 ICMs, Xist transcription was detectable visualization of general transcription within the Xist RNA domain. exclusively from the Xp (Fig. 2A). At mid-stage, Xist transcription It has previously been shown that the X-chromosome is devoid of was maintained on the Xp (Fig. 2A) consistent with Xp-Xist Cot-1 hybridization when gene silencing is established (Chaumeil coating, which was evident by RNA FISH (Fig. 1C). We next et al., 2006; Namekawa et al., 2010). Consistent with predominant quantified the expression of maternal and paternal alleles for four Xp-silencing, we detected mutually exclusive Xist and Cot-1 RNA X-linked genes (Ube1x, Rnf12, Ddx3x and Pdha1), which were FISH signals in the majority (~82%) of trophectoderm nuclei of previously shown to exhibit imprinted Xp silencing in extra- mid-stage blastocysts (Fig. 3A). By contrast, when we performed embryonic tissues (Kalantry et al., 2009). All four X-linked genes RNA FISH on mid-stage ICMs, we detected nuclei with overlap of exhibited biased Xm expression in early ICMs (Fig. 2A). For Cot-1 and Xist RNA FISH signals (Fig. 3B; see Fig. S3 in the Ube1x and Rnf12, very little Xp expression could be detected in supplementary material). Cot-1 RNA FISH signal typically early ICMs. Paternal silencing of Pdha1 and Ddx3x was less overlapped the exterior of the intense Xist domain, although nuclei complete but still favored expression from the Xm. These data were also detected that exhibited an Xp-Xist domain that agree with our RNA FISH results and verify that imprinted XCI is completely overlapped with the Cot-1 RNA FISH signal (see Fig. present in early stage ICMs. By contrast, when individual mid- S3 in the supplementary material). We quantified the number of stage ICMs were analyzed, robust expression from the paternal nuclei per mid-stage ICM that exhibited patterns of mutually allele was detected (Fig. 2A). Ube1x, Rnf12, Pdha1 and Ddx3x all exclusive or overlapping RNA FISH signals (Fig. 3A). On average, exhibited a statistically significant increase in paternal X-linked 45% of nuclei per mid-stage ICM exhibited overlapping signals of

gene expression in mid-stage compared with early ICMs (P<0.05; Cot-1 and Xist. Compared with the trophectoderm, the ICM is, DEVELOPMENT X reactivation precedes loss of Xist RESEARCH ARTICLE 2053

expression pattern of Nanog and Gata6 to be utilized for lineage markers. We found that Nanog and Gata6 nascent transcripts in mid-stage ICMs were largely detected as mutually exclusive cell populations (see Fig. S4B in the supplementary material). On average, ~47% of cells per ICM exhibited exclusive Nanog expression. By contrast, ~32% of cells exhibited exclusive Gata6 expression. On average, 21% of cells per ICM co-expressed both lineage markers (see Fig. S4B in the supplementary material). The degree of mutually exclusive lineage marker expression appears to depend upon the maturity of the blastocyst with more developed blastocysts exhibiting fewer cells with co-expression. These data correspond to predicted patterns of Nanog and Gata6 expression, based primarily on localization at later stages of ICM development, and validate the use of RNA FISH to measure Nanog expression in mid-stage ICMs (Plusa et al., 2008). Restriction of Nanog expression to epiblast progenitors appeared to be more complete in further developed mid-stage blastocysts. Therefore, in the following experiments, we limited our assays to isolated ICMs from blastocysts of ~58-64 cells. In these ICMs, we assayed Nanog expression in cells that exhibited biallelic expression of X-linked genes Atp7a and Mecp2. We found that most cells expressing Atp7a and Mecp2 from both X-chromosomes Fig. 3. Mid-stage inner cell masses (ICMs) exhibit transcription also expressed Nanog (Fig. 4A; see Fig. S4C in the supplementary within the Xp-Xist domain. Cot-1 DNA (red) and Xist (green) probes were used in RNA FISH experiments to determine general Xp material). The reverse was true for Gata6 expression, which, when transcriptional status. (A)The average distribution of nuclei with assayed in independent experiments, was rarely detected in cells mutually excluded or overlapping Cot-1 and Xist RNA FISH signals are exhibiting biallelic X-linked gene expression (see Fig. S4D in the shown as a percentage of total nuclei per mid-stage trophectoderm supplementary material). We also show that in biallelic mid-stage (TE), early ICM and mid-stage ICM. The bracket represents a statistically Nanog-positive cells, Xist clearly coated the Xp (Fig. 4B,C). significant (P

primary transcripts by RNA FISH might confer a more accurate transduction, which has been shown to regulate Nanog expression DEVELOPMENT 2054 RESEARCH ARTICLE Development 138 (10)

Fig. 4. Nanog expression corresponds to biallelic X-linked gene expression in mid-stage inner cell masses (ICMs). (A)Side-by-side box plots graphically represent the percentage of biallelic nuclei (y-axis) grouped as either positive or negative for Nanog transcripts (x-axis). The box plot on the left represents ICMs assayed for Atp7a expression and the right-hand box plot represents ICMs assayed for Mecp2 expression. The medians of the data are indicated (green triangle). The boxes contain the middle 50% of the data. Whiskers represent maximum and minimum values of the dataset. The box plots are nonoverlapping, illustrating that the population of biallelic nuclei are largely skewed towards being Nanog positive. (B)Representative nuclei of a mid-stage ICM showing detection of Atp7a (red), Xist RNA (blue) and Nanog (green) assayed by RNA FISH. Top row: Biallelic Atp7a (red) expression is marked by arrowheads (first panel) with one allele overlapping Xist transcripts (blue, second panel). Nanog transcripts (green) are marked by arrows (third panel). Bottom row: Monoallelic Atp7a expression is marked by an arrowhead (first panel) and colocalizes with Tsix expression (blue pinpoint signal, second panel) from the Xm. In this nucleus, Nanog is not expressed (third panel). (C)The graph represents the distribution of the percentage of nuclei from mid-stage Nanog positive nuclei that exhibit Xp-Xist coating. The red line represents the average. n, the number of embryos analyzed.

(Hamazaki et al., 2006; Pawson and Scott, 1997). NANOG Xp-linked gene activity, we found similar patterns of monoallelic expression was highly variable in the mid-stage ICM, with strong and biallelic Xp-linked gene expression in female Grb2–/– ICMs NANOG immunostaining detectable in only a subset of ICM cells compared with female littermate controls, irrespective of the (Fig. 5A; see Fig. S4A in the supplementary material). By contrast, amount of Xist coating the Xp (Fig. 5C). In fact, for the four genes Grb2–/– blastocysts exhibited strong NANOG immunostaining in we analyzed (Ube1x, Rnf12, Atp7a and Abcb7) monoallelic X- the majority of ICM cells (Fig. 5A) (Chazaud et al., 2006). It has linked gene expression was detectable in cells without visible Xp- been reported that NANOG directly represses Xist expression in Xist coating (Fig. 5B,C). Despite widespread loss of Xp-Xist and undifferentiated mouse ES cells (Navarro et al., 2008). In support Xp-EED accumulation, reactivation in Grb2–/– ICMs occurs of these results, upregulation of Nanog due to inhibition of normally. Analysis of ICM nuclei from late-stage blastocysts (~128 MAPK/ERK signaling resulted in loss of the Xp EED domain cells) also indicates that in the absence of Xist coating, Xp silencing in all ICM cells at E4.5 (Nichols et al., 2009). We also observed can be maintained through cell divisions (data not shown). These that female mid-stage E3.5 Grb2–/– blastocysts exhibited uniform data rule out the possibility that the reactivation detected in the loss of EED and H3K27me3 Xp enrichment specifically in the mid-stage ICM is due to subtle decreases in the levels of Xist and ICM (Fig. 5A; see Fig. S5 in the supplementary material). show that genic silencing in the ICM can be maintained Trophectoderm cells appeared to maintain both inactive-X markers independently of cytologically detectable Xist coating. in wild-type and Grb2–/– blastocysts (Fig. 5A; see Fig. S5 in the supplementary material). We also found a significant reduction in DISCUSSION the number of ICM cells with Xp-Xist coating in Grb2–/– ICMs Here, we present evidence that reversal of imprinted XCI, as compared with littermate controls (Fig. 5B). This was in contrast evident by an increase in Xp-linked gene activity, initiates in the to Grb2–/– embryos isolated between the 8- and 16-cell stage, mid-stage ICM coincident with specification of the epiblast which exhibited normal initiation of Xp silencing and Xp-Xist progenitor population. Genic reactivation occurs in the presence of coating. In addition, we noted that even when Xp-Xist coating was Xist coating, as well as the cytologically detectable PRC2 and detectable in Grb2–/– mid-stage ICMs, the density of the coat was H3K27me3 domains. Together, these observations suggest that,

significantly reduced (Fig. 5C). Importantly, when we analyzed particularly at this early stage of embryonic development, these DEVELOPMENT X reactivation precedes loss of Xist RESEARCH ARTICLE 2055

Fig. 5. Grb2–/– inner cell mass (ICM) cells exhibit loss of Xist transcripts and EED on the Xp. (A)Mid-stage blastocysts immunostained for NANOG (red, top row) and EED (green, second row). NANOG marks the epiblast progenitors. In wild-type blastocysts (left panels), EED accumulates on the Xp in NANOG positive nuclei as seen by the green foci (merge, third row). However, in all but one NANOG positive nuclei of Grb2–/– blastocysts (right-hand panels), Xp EED foci are undetectable (merge, third row). DAPI (blue, fourth row) stains all nuclei. (B)Wild-type (WT, top row) and Grb2–/– (bottom row) mid-stage ICMs assayed by RNA FISH for transcripts of Xist/Tsix (green, left-hand panels) and Ube1x (red, middle panels). Grb2–/– ICMs exhibit a decrease in Xp-Xist coating (green, left panel). A representative nucleus is highlighted and an arrowhead marks monoallelic Ube1x expression, which occurs despite the absence of Xp-Xist coating. The graph indicates the average percentage of cells per ICM that exhibit Xist coating in Grb2–/– compared with wild-type littermate controls. Error bars represent xxx. (C)Nuclei from wild-type (upper rows) and Grb2–/– (bottom rows) ICMs assayed for transcripts of X-linked genes Rnf12, Atp7a and Abcb7 (red signals marked by arrowheads, left panels) and Xist/Tsix (green, middle panels). Grb2–/– ICM nuclei (bottom rows) show monoallelic expression in the absence of the Xp-Xist domain (merge, right panels). The graph indicates the distribution of biallelic X-linked gene expression in Grb2–/– ICMs compared with wild type. The data are presented as a percentage of total cells per ICM. Error bars represent s.d. epigenetic marks, classically analyzed at the cytological level to genes analyzed were chosen at random and are located along the generalize X-chromosome activity, do not accurately reflect the length of the X-chromosome, yet all genes exhibited reactivation expression state of individual X-linked genes. in the presence of Xist or PRC2 accumulation. We argue it is In addition, we argue that genic reactivation is not simply a unlikely that these genes represent a subset of X-linked genes that consequence of loss of Xist coating. We present evidence that X- exhibit a propensity to reactivate. linked gene silencing, at least for a subset of genes, can be Recent data have shown that Xist transcripts first concentrate near maintained even after Xist coating and PRC2 accumulation are the Xist locus, located within the X-chromosome inactivation center removed from the Xp. These data appear to be fundamentally (XIC), and then coating spreads to the rest of the chromosome at odds with the prevailing mechanism for X-chromosome (Sarma et al., 2010); however, how spreading occurs is poorly reactivation, which suggests that reactivation initiates owing to loss understood. It is possible that Xist does not coat the entire X- of these epigenetic modifications. chromosome in a linear fashion. Xist transcripts might favor certain It has long been thought that gene silencing occurs first at X- DNA sequences defined as booster elements (Gartler and Riggs, linked genes located nearest the Xist locus, extends linearly to 1983). Xist transcripts can associate with autosomal DNA distant regions of the X-chromosome, and depends on the spread sequences, suggesting that Xist coating is not dependent on unique of Xist coating (Huynh and Lee, 2003). The ability to analyze X-linked gene sequences. This led to the hypothesis that repetitive imprinted Xp silencing at the level of single cells has now revealed sequences might function as booster elements (Lyon, 1998). A diverse silencing kinetics between genes and no correlation possible explanation for ICM reactivation in the presence of Xist between the proximity to Xist and the speed or efficiency of coating is that, even though Xist, PRC2 and H3K27me3 are detected silencing could be made (Patrat et al., 2009). Although our study cytologically accumulating on the Xp, the spread of these epigenetic analyzes a subset of X-linked genes, the proximity to Xist had no modifications could be incomplete. In both early and mid-stage

consequence in the propensity to reactivate. The seven X-linked ICMs, Xist coating and PRC2 and H3K27me3 might not have DEVELOPMENT 2056 RESEARCH ARTICLE Development 138 (10) reached all genic loci. In the absence of epigenetic modifications at Acknowledgements individual loci, there exists no block to prevent gene specific We thank members of the Magnuson Lab for critically reading the manuscript. reactivation from occurring prior to the chromosome-wide removal This work was supported by a developmental biology training grant graduate fellowship awarded to L.H.W., a K99 postdoctoral award to S.K. and a R01 of epigenetic modifications. Owing to the small number of cells and award to T.M., all from NICHD. Deposited in PMC for release after 12 months. heterogeneity in the developing ICM, we are limited to cytological detection of these epigenetic modifications, which is low resolution Competing interests statement compared with the level of DNA sequence. In the future, analysis The authors declare no competing financial interests. of Xist coating and chromatin modifications at high resolution might Author contributions allow us to determine how these modifications spread to genic loci L.H.W., S.K. and T.M. conceived the study and designed the experimental during pre-implantation development. strategy. L.H.W. conducted the experiments. L.H.W., S.K. and T.M. analyzed The inactive-X in interphase nuclei is organized into a unique the data with support from J.S. for statistical analysis of the RNA FISH data. heterochromatic structure referred to as the Barr body (Barr and L.H.W. wrote the paper; S.K., J.S. and T.M. edited the paper. All authors Carr, 1962). The Barr body can be detected cytologically as a RNA discussed the results and commented on the manuscript. polymerase II-depleted domain, with X-linked genes positioned on Supplementary material the outer rim of an interior core composed primarily of repetitive Supplementary material for this article is available at non-coding sequences (Chaumeil et al., 2006; Clemson et al., http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.061176/-/DC1 2006). Examination of XCI kinetics suggests that formation of the silent, repetitive non-coding core occurs prior to X-linked gene References Agulnik, A. I., Mitchell, M. J., Mattei, M. G., Borsani, G., Avner, P. A., Lerner, silencing (Chaumeil et al., 2006; Namekawa et al., 2010). Thus, it J. L. and Bishop, C. E. (1994). A novel X gene with a widely transcribed Y- has been proposed that repression of repetitive non-coding linked homologue escapes X-inactivation in mouse and human. Hum. 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